Competitive Reporter Monitored Amplification (CMA) - Quantification of Molecular Targets by Real Time Monitoring of Competitive Reporter Hybridization

Background: State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests. Methodology and Principal Findings: The described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique. Conclusions and Significance: The system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution fo

Routine use of a highly automated and internally controlled real-time PCR assay for the diagnosis of herpes simplex and varicella-zoster virus infections

BACKGROUND: Detection of herpes viruses can be significantly improved by PCR. The development of real-time PCR, which has overcome several limitations of conventional PCR, improved the prospects for implementation of PCR-based assays in diagnostic laboratory. OBJECTIVES: To compare the diagnostic performance of an automated sample extraction procedure in combination with an internally controlled real-time PCR assay for detection of herpes simplex virus (HSV) and varicella-zoster virus (VZV) to conventional shell vial culture. STUDY DESIGN: One hundred eighty-two consecutive specimens from patients suspected of HSV or VZV infection were examined by internally controlled PCR and shell vial culture. An internal control consisting of phocine herpes virus was processed along with the specimens during the entire procedure and permitted to monitor extraction and amplification efficiency, including inhibition. RESULTS: A total of 48 (26.4%) specimens were positive for HSV or VZV by culture, and 77 (42.3%) by real-time PCR. Thus, overall sensitivity increased by 60.4%. All culture-positive specimens were detected and typed correctly by PCR, except for a single specimen that contained PCR inhibitors. Specifically, the real-time PCR assay increased the detection rate for HSV-1 and HSV-2 by 43.9% and 62.5%, respectively. In PCR-positive specimens, lower levels of viral DNA were found in culture-negative than in culture-positive specimens. The increase of HSV detection rates by PCR varied with the origin of specimen and was particularly significant for skin specimens (7/14 versus 3/14 detected by culture) and bronchoalveolar lavages (8/8 versus 1/8). In addition, real-time PCR significantly increased the detection rate for VZV. CONCLUSIONS: Compared to shell vial culture, our real-time PCR assay demonstrated a superior sensitivity and an added value of using internal control for checking the quality of examination of each specimen. These results provide a solid basis for implementation of real-time PCR in the routine diagnosis of HSV and VZV infections in various clinical specimens.status: publishe

Application of UL144 Molecular Typing To Determine Epidemiology of Cytomegalovirus Infections in Preterm Infants

Sequence analysis of the UL144 gene of human cytomegalovirus (CMV) was used to investigate the epidemiology of CMV infections in preterm infants. Nosocomial transmission of CMV from congenitally infected infant to preterm twins was excluded based on distinct molecular profiles of CMV strains. Indistinguishable molecular profiles between strains from the mother and the infant indicated postnatal acquisition of CMV through breastfeeding

Spectrum of antiviral activity of o-(acetoxyphenyl)hept-2-ynyl sulphide (APHS).

Contains fulltext : 47348.pdf (publisher's version ) (Closed access)Since some antiviral drugs have a broad spectrum of action, the aim of this study was to assess whether o-(acetoxyphenyl)hept-2-ynyl sulphide (APHS), a recently described inhibitor of human immunodeficiency virus type 1 (HIV-1) replication, has an effect on the replication of other retroviruses, (-) and (+) RNA viruses and DNA viruses. APHS did not affect the replication of feline immunodeficiency virus, HIV-2 and a HIV-1 strain resistant to non-nucleoside reverse transcriptase inhibitors (NNRTI). APHS could also not inhibit the replication of the RNA viruses, respiratory syncytium virus or mouse hepatitis virus. In contrast, APHS did inhibit the replication of wild-type herpes simplex virus type 1 (HSV-1) as well as acyclovir-resistant HSV-1 and HSV-2 mutant. These results suggest that APHS is a NNRTI of HIV-1 replication, but not HIV-2 replication, and that APHS is an inhibitor of both HSV-1 and HSV-2 replication

ELVIRA HSV, a yield reduction assay for rapid herpes simplex virus susceptibility testing

A colorimetric yield reduction assay, ELVIRA (enzyme-linked virus inhibitor reporter assay) HSV, was developed to determine the antiviral drug susceptibilities of herpes simplex virus (HSV). It uses an HSV-inducible reporter cell line. This simple and rapid assay has an objective readout, low inoculum size, and good reproducibility. The results correlate well with those of the plaque reduction assay.status: publishe

Survey of acyclovir-resistant herpes simplex virus in the Netherlands: prevalence and characterization

BACKGROUND: Widespread and frequent use of acyclovir (ACV) for treatment, suppressive therapy and prophylaxis of herpes simplex virus (HSV) infections and its over the counter availability may be associated with emergence of HSV resistance. OBJECTIVES: To determine the prevalence of ACV-resistant HSV isolates in different patient groups between 1999 and 2002 in the Netherlands. STUDY DESIGN: A total of 542 isolates, 410 HSV-1 and 132 HSV-2, from 496 patients were screened for reduced susceptibility to ACV. A newly developed ELVIRA HSV screening assay was used that allowed a high throughput screening. The genotypic analysis of the HSV thymidine kinase gene was performed to identify resistance-associated mutations. RESULTS: Thirteen isolates, 8 HSV-1 and 5 HSV-2, from 10 patients (2%) were found resistant to ACV. A single ACV-resistant strain was identified among isolates from 368 immunocompetent patients (0.27%; 95% confidence interval [CI], 0.007%-1.5%), whereas in nine isolates from 128 immunocompromised patients resistant HSV was identified (7%; 95% CI, 3.26%-12.93%). The highest frequency of ACV-resistant HSV was associated with bone marrow transplantation: four patients out of 28 (14.3%) shed resistant virus. In addition, resistant virus was obtained from two HIV-positive patients, one patient with a hematological malignancy and two patients on immunosuppressive drugs. Further testing showed that none of the isolates was resistant to foscarnet. Several new mutations were identified in the thymidine kinase gene of these resistant isolates, and their effect on ACV-resistance is discussed. CONCLUSIONS: Our study shows that the prevalence of ACV resistance is low in immunocompetent patients (0.27%), whereas ACV-resistant HSV infections occur relatively frequently in immunocompromised patients (7%; P < 0.0001). This emphasizes the need for drug susceptibility monitoring of HSV infections in immunocompromised patients with persisting infections despite antiviral therapy.status: publishe

Developments in mycotoxin analysis: an update for 2020-2021

This review summarises developments published in the period from mid-2020 to mid-2021 on the analysis of a number of diverse matrices for mycotoxins. Notable developments in all aspects of mycotoxin analysis, from sampling and quality assurance/quality control of analytical results, to the various detection and quantitation technologies ranging from single mycotoxin biosensors to comprehensive instrumental methods are presented and discussed. The summary and discussion of this past year's developments in detection and quantitation technology covers chromatography with targeted or non-targeted high resolution mass spectrometry, tandem mass spectrometry, detection other than mass spectrometry, biosensors, as well as assays using alternatives to antibodies. This critical review aims to briefly present the most important recent developments and trends in mycotoxin determination, as well as to address limitations of the presented methodologies

Developments in mycotoxin analysis: An update for 2017-2018

This review summarises developments that have been published in the period from mid-2017 to mid-2018 on the analysis of various matrices for mycotoxins. Analytical methods to determine aflatoxins, Alternaria toxins, ergot alkaloids, fumonisins, ochratoxins, patulin, trichothecenes, and zearalenone are covered in individual sections. Advances in sampling strategies are discussed in a dedicated section, as are methods used to analyse botanicals and spices, and newly developed comprehensive liquid chromatographic-mass spectrometric based multi-mycotoxin methods. This critical review aims to briefly discuss the most important recent developments and trends in mycotoxin determination as well as to address limitations of the presented methodologies